The present invention relates to plant genetic engineering methods useful for the alteration of the constituents of plant seeds. More specifically, the invention relates to promoters that have been obtained from flax and are capable of directing expression of non-native genes in flax seeds as well as the seeds of other plants.
Flax or linseed (Linum usitatissimum) is a commercially important oilseed crop. Flax oil and meal are valuable raw materials derived from flax seed. A further economically significant raw material, flax fiber, is obtainable from the stem of the plant. The flax oil fraction is used for non-edible purposes, for example in the manufacture of varnish and paint, and has more recently become suited for use in the manufacture of a range of edible products, such as margarines and salad oils and dressings, by virtue of newly bred so called Linola cultivars (Green (1986) Can. J. Plant Sci, 66: 499-503). Flax meal is used primarily as a constituent of ruminant feeds while flax fibers are used in the manufacture of linen fabrics. Given its economic importance as a source for raw materials, it is desirable to further improve and diversify the available flax cultivar portfolio both with respect to agronomic performance, for example seed yield, resistance to pathogens and low climatic temperatures, and with respect to yield and quality of the raw materials to suit downstream applications. Although it is possible to obtain improved flax cultivars through conventional plant breeding, as evidenced by the development of the Linola cultivars, developing an elite agronomic plant line requires large investments in plant breeding due to the long timelines involved. Plant genetic engineering technology allows the isolation of genes directly from unrelated species and the transfer of these genes into elite agronomic backgrounds, thereby significantly reducing the time required to develop new cultivars. In addition plant genetic engineering permits the manufacture of products not naturally obtainable from flax, for example therapeutic agents.
In order to develop novel flax cultivars through plant genetic engineering, control over the expression of the introduced foreign or non-native gene is of critical importance. The desired expression characteristics for the non-native gene, such as the level of expression of the non-native gene, the particular plant tissue or organ in which the non-native gene is expressed, and the particular time in the growth cycle of the plant at which the non-native gene is expressed, will vary depending on the application for which the plant line is developed. For example, the modification of the seed oil composition may require low levels of seed-specific expression of an enzyme involved in fatty acid metabolism at an early stage in seed development (see for example U.S. Pat. No. 5,420,034). On the other hand expression of a pharmaceutical protein could preferably require high levels of leaf-specific expression upon harvesting of the plant leaves (see for example, U.S. Pat. No. 5,929,304).
In order to manipulate the expression characteristics of non-native genes numerous factors can be influenced. One factor is the choice of the transcriptional promoter used. A wide range of plant compatible promoters is currently available and some of the better documented promoters include constitutive promoters such as the 35S CaMV promoter (Rothstein et al. (1987), Gene 53: 153-161) and the ubiquitin promoter (U.S. Pat. No. 5,614,399), tissue specific promoters such as seed-specific promoters, for example the phaseolin promoter (Sengupta-Gopalan et al., (1985), PNAS USA 82: 3320-3324) and inducible promoters, such as those inducible by heat (Czarnencka et al., (1989), Mol. Cell. Biol. 9 (8): 3457-3464), UV light, elicitors and wounding (Lois et al., (1989) EMBO J. 8 (6): 1641-1648), or chemicals such as endogenous hormones (Skriver et al. (1991), Proc. Natl. Acad. Sci. USA 88(16): 7266-7270). Other factors that can be manipulated in order to control the expression characteristics of non-native gene in plants include transcriptional modification factors such as introns, polyadenylation sites and transcription termination sites. The expression characteristics of the non-native gene can further be manipulated by factors that affect translation, such as ribosomal binding sites and the codon bias that is exhibited by the host. Furthermore, the non-native gene itself may affect the viability of the transgenic plant, thus limiting particularly the levels of expression that can be attained. In some cases it may be possible to overcome this problem, by expressing the protein in a tissue specific manner, e.g. in the leaves or seed, or by restricting the accumulation of the protein in different subcellular compartments such as for example the cytoplasm, the endoplasmic reticulum or vacuoles, typically by the presence or the absence of specific targeting sequences capable of directing the protein to these compartments. Another factor that will affect the expression characteristics is the location in which the construct inserts itself into the host chromosome. This effect could provide an explanation as to why different plants, transformed with the same recombinant construct, can have fluctuating levels of recombinant protein expression.
To the best of the inventors"" knowledge, expression of non-native genes in flax seeds is only documented in PCT Patent Aplication WO 98/18948. This application discloses two stearoyl-acyl carrier protein desaturase (SAD) genes derived from flax. The associated SAD promoter sequences are useful for the modification of flax and other plants for the expression of endogenous or foreign genes. However the methods taught by WO 98/18948 are limited by the fact that the SAD promoters are not seed-specific in flax and confer expression to leaves, stems, flowers and seeds. Expression of non-native genes thus may result in undesirable side effects in non-seed tissues. In addition the use of the SAD promoters allows limited control over expression level and timing of expression.
There is a need in the art to further improve methods for the expression of non-native genes in flax seeds and other plant seeds.
The present invention relates to improved methods for the seed-specific expression of non-native genes in plants. In particular, the invention relates to improved methods for the seed-specific expression of non-native genes in flax.
Accordingly, in one aspect, the invention provides a method for the expression of a nucleic acid sequence of interest in flax seeds comprising:
(a) preparing a chimeric nucleic acid construct comprising in the 5xe2x80x2 to 3xe2x80x2 direction of transcription as operably linked components
(1) a seed-specific promoter obtained from flax; and
(2) the nucleic acid sequence of interest wherein said nucleic acid of interest is non-native to said flax seed-specific promoter;
(b) introducing said chimeric nucleic acid construct into a flax plant cell; and
(c) growing said flax plant cell into a mature flax plant capable of setting seed, wherein said nucleic acid sequence of interest is expressed in the seed under the control of said flax seed-specific promoter.
In a preferred embodiment of the invention, at least one expression characteristic, e.g. timing of expression in the plant""s life cycle, conferred by the promoter to the non-native nucleic acid sequence is similar to that expression characteristic when conferred to a native nucleic acid sequence. In further preferred embodiments, the flax seed-specific promoter is an oleosin promoter, a 2S storage protein promoter or a legumin-like seed storage protein promoter.
In a further aspect, the present invention provides transgenic flax seeds prepared according to a method comprising:
(a) preparing a chimeric nucleic acid construct comprising in the 5xe2x80x2 to 3xe2x80x2 direction of transcription as operably linked components:
(1) a seed-specific promoter obtained from flax; and
(2) a nucleic acid sequence of interest wherein said nucleic acid of interest is non-native to said seed-specific promoter;
(b) introducing said chimeric, nucleic acid construct into a flax plant cell; and
(c) growing said flax plant cell into a mature flax plant capable of setting seed, wherein said nucleic acid sequence of interest is expressed in the seed under the control of said seed-specific promoter.
In a further aspect the present invention provides flax plants capable of setting seed prepared by a method comprising:
(a) preparing a chimeric nucleic acid construct comprising in the 5xe2x80x2 to 3xe2x80x2 direction of transcription as operably linked components:
(1) a seed-specific promoter obtained from flax; and
(2) a nucleic acid sequence of interest wherein said nucleic acid of interest is non-native to said seed-specific promoter;
(b) introducing said chimeric nucleic acid construct into a flax plant cell; and
(c) growing said flax plant cell into a mature flax plant capable of setting seed, wherein said nucleic acid sequence of interest is expressed in the seed under the control of said seed-specific promoter.
In yet a further aspect, the present invention provides novel flax seed specific promoters useful for the expression of non-native genes in flax seeds and the seeds of other plant species, useful for example for modification of the protein or oil composition of the seed.
In a preferred embodiment, the seed specific promoter comprises:
(a) a nucleic acid sequence as shown in FIG. 1 (SEQ.ID.NO.:1), FIG. 2 (SEQ.ID.NO.:4), FIG. 3 (SEQ.ID.NO.:6) or FIG. 4 (SEQ.ID.NO.:8) wherein T can also be U;
(b) a nucleic acid sequence that is complimentary to a nucleic acid sequence of (a);
(c) a nucleic acid sequence that has substantial sequence homology to a nucleic acid sequence of (a) or (b);
(d) a nucleic acid sequence that is an analog of a nucleic acid sequence of (a), (b) or (c); or
(e) a nucleic acid sequence that hybridizes to a nucleic acid sequence of (a), (b), (c) or (d) under stringent hybridization conditions.
In another aspect, the invention provides chimeric nucleic acid sequences comprising a first nucleic acid sequence obtained from flax operatively linked to a second nucleic acid sequence non-native to said first nucleic acid sequence wherein said first nucleic acid sequence comprises a novel flax seed-specific promoter.
Other features and advantages of the present invention will become readily apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples while indicating preferred embodiments of the invention are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art of this detailed description.